Progress 05/01/24 to 04/30/25
Outputs
Target Audience:This project actively engaged scientists and students, including individuals from underrepresented groups in plant and insect sciences. Preliminary findings focusing on the nutritional profiles of western corn rootwormon maize were presented as a poster at the Entomological Society of America (ESA) Annual Meeting in November 2024 in Phoenix, AZ. More recently,an abstract highlighting recent findings on host specialization and nutritional adaptation in western corn rootworm has been submitted for an oral presentation at the upcoming ESA Annual Meeting in November 2025 in Portland, OR. A manuscript describingthefindings of this project is currently in preparation and will be submitted for peer-reviewed publication shortly. Once published, it will be made publicly available as an open-access article. Additionally, the findings will be shared with the broader public through the University of Missouri's outreach platforms. Preliminary outcomes have also been communicated to industry researchers through direct conversations between the principal investigator (Huynh) and scientific collaborators from private-sector partners, including NewLeaf Symbiotics and Frontier Agricultural Sciences. Changes/Problems:One of the primary challenges encountered during the project was the complexity of extended greenhouse experiments, each requiring 3-4 months to complete, which involved rearing of western corn rootwormneonates on maize plants under greenhouse conditions. These experiments were expected to be challenging, but the vulnerability of insect neonatesto environmental fluctuations (e.g., drought, overheating) posed additional unforeseen obstacles. As a result, initial experiments yielded an insufficient number of viable insect samples. To address this issue, we conducted a series ofgreenhouse experiments to ensure the collection of healthy insect samples for metabolomics analyses. While this significantly extended the project timeline, it was essential to ensure the scientific quality and meaningful impact of the project's outcomes. Despite this delay, substantial progress has been made toward achieving the project's goals. To ensure successful execution within the final project year, we recruited a full-time research scientist (1.0 FTE) with expertise in analytical chemistry and metabolomics. This strategic addition is expediting metabolomics and chemical analyses and supporting the timely completion of remaining project components, including insect functional bioassays, bioinformatics integration, and preparation of the final report and publications. What opportunities for training and professional development has the project provided?Greenhouse and laboratory experiments investigating plant and insect biology have been conducted. Two research assistants participated in these research activities. Both received training in conducting plant and insect research under greenhouse and laboratory conditions. A research scientist attended and presented the project findings at theAnnual Meetingof the Entomological Society of America in November 2024 in Phoenix, AZ. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?I anticipate completing the project goalsduring thefinal project year. We will determine the chemical nutritional composition of maize and non-maize host plants and integrate these plant nutritional profileswith insect metabolic profilesto identify candidate nutrients for functional validation through insect diet assays. These insectbioassays will be designedas mixture experiments to evaluate both individual and interactive effects of selected candidate nutrients (evaluating up to eight nutrients per experiment) on selective insectlife history parameters, including larval survival, molting, weight, pupation, and adult emergence.Experimental designs will be generatedusing Design-Expertsoftware (v.10.0, Stat-Ease, Inc., Minneapolis, MN), and response surface models will be generated to describe the relationships between nutrient combinations and insect development. Model selection will be based on key statistical criteria, including lack of aliased terms, significant model P-values, high R² values, and low predicted residual error sum of squares (PRESS). Through this approach, we expect to identify key nutrients and their optimal combinations that support larval development and successful pupation on refined diets, comparable to those reared on natural maize roots.
Impacts
What was accomplished under these goals?
Objective 1: Identify nutritional requirements of the western corn rootworm to successfully develop to adulthood. To identify the specific nutrients required for successful pupation of the western corn rootworm (WCR),we conducted greenhouse experiments to generate both maize root and insect tissue samples for downstream metabolomic and nutritional analyses. Experiments were conducted using a non-transgenic maize line (Viking 60-01N, Albert Lea Seed) grown in 19-liter pots under greenhouse conditions. Approximately 100,000 WCR eggs from a lab-reared, non-diapausing strain maintained at USDA-ARS (Columbia, MO) were used to infest plants. Neonate larvae (<24 hours old) were infested at a densityof 60 per pot across four maize growth stages (V2, V7, V18, R2) to generate sufficient insect samples. Larvae and pupae were manually collected at 17 and 21 days post-infestation, corresponding to the 3rd instar and pupal stages. Insect samples were collected from four biological replicates per stage, each consisting of 10-30 individuals (50-100 mg), flash-frozen in liquid nitrogen, and stored at -80°C for metabolomics analysis. Insect tissues were extracted with methanol and analyzed by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using four analytical platforms: two reverse-phase (RP)/UPLC-MS/MS methods in positive ion mode electrospray ionization (ESI), one RP/UPLC-MS/MS method in negative ion mode ESI, and one hydrophilic interaction liquid chromatography (HILIC)/UPLC-MS/MS method in negative ion mode ESI. These analyses identified over 600 metabolites, classified into biochemical categories including amino acids, carbohydrates, lipids, energy metabolites, nucleotides, cofactors, vitamins, and plant secondary metabolites. Multivariate statistical analysis and pathway enrichment analysis were utilized to identify candidate nutrients and nutritional pathways for further functional characterization. Simultaneously, maize root tissues from the same phenological stages (V2, V7, V18, and R2) were collected from uninfested plants and are being analyzed for their comprehensive nutritional composition, including complete amino acid profiles, soluble sugars, carbohydrates, crude protein, and crude fiber. These root nutrient profiles will be integrated with WCR metabolomics data to identify key biofactors involved in larval development and pupation. Diet bioassays will be conducted using response surface modeling integrated with mixture experiments to determine the impact of these nutrients on WCR development. Objective 2: Determine nutritional factors limiting the western corn rootworm development on non-maize hosts. To identify the specific nutrients limiting WCR development on non-maize hosts, we conducted a series of greenhouse experiments to generate sufficient insect tissue samples from larvae, pupae, and adults reared on non-maize host and non-host plants, including wheat, oat, and wheatgrass. Seeds of each plant species were sown in 10-liter pots filled with a planting medium and maintained under greenhouse conditions. Approximately 400 WCR eggs were introduced into each pot, with 40 replicate pots prepared per plant species to generate insect samples. After 10 days post-infestation, pots were monitored daily to collect insect samples. Third instar, pupae, and adults were manually collected for subsequent analyses as described above. These insect samples were utilized for global metabolomics analysis as described above. Over 500 metabolites and associated metabolic pathways were identified, providing insight into potential nutritional limitations affecting WCR development on non-maize host plants compared to maize as the natural host. Concurrently, roots of the non-maize plants are being analyzed for their chemicalnutritional composition. These root nutrient profiles will be integrated with the insect metabolomics data to identify key nutrient factors limiting WCR development on non-maize hosts. These nutrient factors will be functionally characterized using insect diet bioassays. During the first year of the project, we developed an improved artificial diet formulation and bioassay technique specifically utilized for WCR development. These advancements enable evaluation of the effects of individual nutrients on WCR pupation and development, based on insect life history parameters (e.g., weight, survival, molting). This bioassay system will be employed to characterize both the individual and combined effects of candidate nutrients using response surface modeling integrated with mixture experiments. These candidates will be selected based on an integrated analysis of WCR metabolomic profiles and the chemical composition of roots from various host and non-host plants, guided by statistical modeling and metabolic pathway enrichment analysis.
Publications
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Progress 05/01/23 to 04/30/24
Outputs
Target Audience:Scientists and students including those from underrepresented groups arein plant sciences:Parts of the findings from this project resulted in an abstract describing the nutritional profiles of western corn rootworms on maize and artificial diets. The abstract has been submitted to be presented at the annual meetings of the Entomological Society ofAmerica in November2024 inPhoenix, AZ. Industry researchers: multiplescientists from different private industries (i.e., New Leaf Symbiotics,Bioconsortia, and Genective)were introduced to brief information on the project via personal communication of PI (Huynh) and co-PI (Hibbard). Whenthe findings from the projects are publicly available, they will be shared with industry researchers. Changes/Problems:Long-time greenhouse experiments (3-4 months) involving the cultivation of maize plants and rearing of insects fed on the plants were anticipated to be challenging. We aimed to collect healthy insects; however, insects are vulnerable to unfavorable conditions such as drought and overheating caused by temperature fluctuations that may occur in the greenhouse during 4-month experiments. As the first experiment did not provide sufficient insect samples, we conducted additional experiments to obtain the necessary samples for analysis, which extended the sample collection period. To accelerate the project, a research scientist (0.2 FTE) has been hired dedicated specifically to metabolomics analysis. What opportunities for training and professional development has the project provided?Greenhouse and laboratory experiments studying plant and insect biology have been conducted. One undergraduate student and two research assistants have participated in these research activities. They have trained to perform research on plants and insects in greenhouse and laboratory conditions. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?I anticipate completing the nutritional profiles of the western corn rootworm larvae as well as the chemical composition analyses on maize and insect diets. Additionally, I expect to finalize the screening of candidate nutrients. We aim to submit at least one paper to a high impact factor journal in Entomology (e.g., Scientific Reports) and at least one presentation (oral talk or poster) will be presented at a national or international scientific conference. Furthermore, we plan to provide training to at least one undergraduate student and one research scientist from underrepresented groups in plant and insect research. We are currently determining the nutritional profiles of the western corn rootworm larvae and conducting chemical composition analyses of maize roots and roots from non-host plants. Global metabolomic analyses will be performed to determine metabolic profiles of the western corn rootworm fed on these roots using ultrahigh-performance liquid chromatography-tandem mass spectroscopy (UHPLC-MS/MS). The chemical composition analyses of the maize roots will be conducted by the Agricultural Experiment Station Chemical Laboratories at the University of Missouri (Columbia, MO) using official analytical methods. These profiles will be examined to identify potential candidate nutrients involved in the western corn rootworm pupation via multivariate analyses and pattern hunter algorithms. The identified candidate nutrients will be further evaluated for their effects on the western corn rootworm larval development and pupation using diet bioassays coupled to response surface modeling. Additionally, metabolic pathways in which these nutrients are expressed differently in the western corn rootworm larvae fed on different plant species will be identified using pathway analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Differences in absolute amounts of key nutrients that enhance the western corn rootworm development and pupation will be quantitated in the western corn rootworm larvae at different developmental stages (2nd and 3rd instars, and pupae) and in roots of different plant species using liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Insect bioassays designed as mixture experiments will be used to concurrently characterize single and combination effects of candidate nutrients (8 nutrients per experiment) on the life history parameters (survival, molting, weight, pupation and adult emergence rates). Experimental designs will be developed using Design-Expert (v.10.0, Stat-Ease, Inc., Minneapolis, MN). Response surface models will be generated to describe the effects of the candidate nutrients on larval survival, weight, molting, and proportions of pupation and adult emergence. Model selection will be based on criteria such as a lack of aliased terms, low model P-value, high R-values, and a low PRESS value. We expect to determine key nutrients and their optimum blends that support the western corn rootworm larval development and pupation on refined diets, comparable to those fed on maize roots.
Impacts
What was accomplished under these goals?
Objective 1: Identify nutritional requirements of the western corn rootworm to successfully develop to adulthood. In the first year of the project, we aimed to perform plant and insect assays to generate maize root samples and insects fed on maize roots, followed by the initiation of the analyses to identify the nutrients that are required for western corn rootworm pupation. Greenhouse experiments were conducted to generate maize root samples and insect samples (whole larvae at different developmental stages (2nd and 3rd instar, prepupae, and pupae) using a non-transgenic maize line (Viking 60-01N, Albert Lea Seed, Albert Lea, MN). Maize was grown in 400 containers (19-liter plastic pots) filled with a planting medium in a greenhouse. The maize plants were infested with the western corn rootworm neonates (~100,000 eggs were used to obtain a sufficient number of larvae) from a non-diapausing strain. This strain, originally established from eggs purchased from Crop Characteristics (Farmington, MN), has been maintained on the non-transgenic maize line (Viking 60-01N, Albert Lea Seed) for multiple generations in the Plant Genetics Research Unit-USDA-ARS (Columbia, MO). Neonate larvae (<24 h old) were infested at a density of 60 neonates per pot across four different maize phenological stages (V2, V7, V18, and R2), with thirty pots prepared for each stage to obtain sufficient numbers of larvae and pupae. At day 7, 17 and 21 post-infestation, larvae that developed to 2nd instar, 3rd instar, and pupae were manually collected from soil and maize roots in the plant containers, respectively. Insect samples were collected from different pots in four replicates for each root phenological stage, with each replicate containing 10-30 insect specimens (or approximately 50-100 mg). The insect samples were immediately flash-frozen in liquid nitrogen and stored at −80 °C for global metabolomics analysis. Concurrently, twenty plant containers per maize phenological stage without larval infestation were prepared to collect root samples. Roots from these plants were collected in four replicates (~200 g per replicate, wet weight basis) from different plant containers. After rinsing with water to remove soil, clean roots were cut into pieces less than 2 cm and immediately frozen at −80 °C for chemical composition analyses. The untargeted metabolomics and chemical composition analyses of the insect and maize root samples aim to determine the metabolic profiles of the western corn rootworm larvae and pupae, and nutritional profiles of maize roots including complete amino acid profiles, sugars, carbohydrates, crude protein, moisture, ash, crude fiber. These profiles will be crucial in identifying the specific nutrients that are required for western corn rootworm pupation. Objective 2: Determinenutritional factors limiting the western corn rootworm development on non-maize hosts. In the first year, our goal was to conduct plant assays to generate insects reared on non-maize host and non-host plants (i.e., wheat, oat, wheatgrass, witch grass) and collect roots of these plant species. We conducted the plant assays and initiated the analyses to determine nutrients that limit the development of the western corn rootworm in these non-host plants. Seeds of each plant species were planted in 10-litter pots filled with a planting medium in a greenhouse. Approximately 400 western corn rootworm eggs were infested in each pot. Forty containers per plant species were prepared to obtain adequate numbers of larvae for designed analyses. Plants were regularly watered as necessary and fertilized every 7 d for 4 weeks. After 10 days, plant containers were checked daily to collect 2nd and 3rd instars or pupae of the western corn rootworm. These insect samples were used for global metabolomics analysis as described above. Additionally, twenty pots per plant species, which were not infested with western corn rootworm eggs, were prepared to collect roots for the chemical composition analyses as described above. The untargeted metabolomics and the chemical composition analyses aim to determine the metabolic profiles of western corn rootworm larvae and pupae and the nutritional profiles of the roots of these plant species. These profiles will be crucial in identifying nutritional factors limiting the western corn rootworm development on non-maize hosts. Moreover, during the first year, we developed a newly, improved diet formulation and bioassay techniques, specifically for the western corn rootworm. These advancements can allow us to differentiate the effects of nutrients on pupation development, based on the life history parameters of the western corn rootworm. This insect bioassay will be utilized to characterize both single and combination effects of candidate nutrients. These candidates will be selected from the nutritional profiles of the western corn rootworm and the chemical composition profiles of roots from various host and non-host plants based on statistical modeling and metabolic pathway analysis.
Publications
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